1. Field of the Invention
This invention relates generally to sensing the presence of magnetic particles, and more particularly to quantitatively measuring accumulations of such particles by means of DC magnetic excitation and sensing of the amplitude of the resulting excitation of the magnetic moments of the particles.
2. Discussion of Related Art
Much attention has been given to techniques for determining the presence, and possibly the level of concentration, of minute particles in a larger mixture or solution in which the particles reside. It is desirable in certain circumstances to measure very low concentrations of certain organic compounds. In medicine, for example, it is very useful to determine the concentration of a given kind of molecule, usually in solution, which either exists naturally in physiological fluids (for example, blood or urine) or which has been introduced into the living system (for example, drugs or contaminants).
One broad approach used to detect the presence of a particular compound of interest is the immunoassay technique, in which detection of a given molecular species, referred to generally as the ligand, is accomplished through the use of a second molecular species, often called the antiligand or the receptor, which specifically binds to the ligand of interest. The presence of the ligand of interest is detected by measuring, or inferring, either directly or indirectly, the extent of binding of ligand to antiligand.
A good discussion of several detection and measurement methods appears in U.S. Pat. 4,537,861 (Elings et al.). The patent is directed to several ways to accomplish homogenous immunoassays in a solution of a binding reaction between a ligand and an antiligand which are typically an antigen and an antibody. The teaching of Elings is to create a spatial pattern formed by a spatial array of separate regions of antiligand material attached to a solid substrate. The corresponding ligand, which has been previously labeled by attaching to it a molecule or particle which has a particular physical characteristic, is then dispersed over the solid substrate such that the labeled ligand can produce a binding reaction with the antiligand in the spatial patterns. After the labeled bound complexes have been accumulated in the spatial patterns, equipment is used to scan the solid substrate, thereby measuring the physical characteristic of the labels to provide the desired immunoassay. The scanner may be based on fluorescence, optical density, light scattering, color and reflectance, among others. In addition, Elings further teaches that the magnetic particles may also be attached to either the ligand or the labeled ligand for the purpose of accumulating the labeled bound complexes within the solution or onto the prepared substrate surface, after which the scanning techniques previously described are employed.
Indeed, magnetic particles made from magnetite and inert matrix material have long been used in the field of biochemistry. They range in size from a few nanometers up to a few microns in diameter and may contain from 15% to 100% magnetite. They are often described as superparamagnetic particles or, in the larger size range, as magnetic beads. The usual methodology is to coat the surface of the particles with some biologically active material which will cause them to bond strongly with specific microscopic objects or particles of interest (proteins, viruses, cells, DNA fragments, for example). The magnetic particles then become xe2x80x9chandlesxe2x80x9d by which the objects can be moved or immobilized using a magnetic gradient, usually provided by a strong permanent magnet. The Elings patent is an example of this use of magnetic particles. Specially constructed fixtures using rare-earth magnets and iron pole pieces are commercially available for this purpose.
Although these magnetic particles have been used primarily for moving or immobilizing the bound objects, some experimental work has been done on using the particles as tags for detecting the presence of the bound complexes. Historically the detection and quantification of the bound complexes has been accomplished by means of radioactive, fluorescent, or phosphorescent molecules which are bound to the complexes of interest. These prior tagging techniques have various important weaknesses. Radioactive methods present health and disposal problems of the resulting low-level radioactive waste, and they are also relatively slow. Fluorescent or phosphorescent techniques are limited in their quantitative accuracy and dynamic range because emitted photons may be absorbed by other materials in the sample (see Japanese patent publication 63-90765, published Apr. 21, 1988, Fujiwara et al.). Furthermore, the signal from the fluorescent or phosphorescent molecules normally decays over a period of hours or perhaps days, at the most.
On the other hand, since the signal from a tiny volume of magnetic particles is exceedingly small, it has been natural that researchers have tried building detectors based on Superconducting Quantum Interference Devices (SQUIDs), which are well known to be the most sensitive detectors of magnetic fields for many applications. There are several substantial difficulties with this approach, however. Since the pickup loops of the SQUID must be maintained at cryogenic temperatures, the sample must be cooled to obtain a very close coupling to these loops. This procedure makes the measurements unacceptably tedious, and is inappropriate for many biotechnology applications. In addition, the general complexity of SQUIDS and their associated cryogenic components renders them extremely expensive and generally unsuitable for use in an inexpensive desktop instrument. Even a design based on xe2x80x9chigh Tcxe2x80x9d superconductors does not completely overcome these objections, and would introduce several new difficulties, as discussed in Fujiwara et al.
More traditional approaches to detecting and quantifying the magnetic particles have typically involved some form of force magnetometry, in which the sample is placed in a strong magnetic gradient and the resulting force on the sample is measured. In a force-balance magnetometer, for example, the force is measured as an apparent change in the weight of the sample as the gradient is changed. An example of this technique is shown in Rohr U.S. Pat. Nos. 5,445,970 and 5,445,971. A more sophisticated technique measures the effect of the particle on the deflection or vibration of a micromachined cantilever (see Baselt et al., A Biosensor based on Force Microscope Technology, Naval Research Lab., J. Vac. Science Tech. B., Vol 14, No. 2, 5pp, April 1996). These approaches are all limited in that they rely on converting an intrinsically magnetic effect into a mechanical response, which must then be distinguished from a large assortment of other mechanical effects such as vibration, viscosity, and buoyancy, which can substantially interfere with the intended measurement.
In U.S. Pat. No. 6,046,585, Simmonds describes a technique employing a small region (the xe2x80x9cgapxe2x80x9d) in a toroidal magnetizer, within which one places a pair (or multiple pairs) of inductive detection coils and generates a high-frequency oscillating magnetic field (the xe2x80x9cdrive fieldxe2x80x9d). In this implementation, the individual detection coils are carefully matched in size but counter-wound, so that in the absence of any other magnetic materials (such as magnetic particles which are part of magnetic bound complexes) the pair of coils produces a zero output voltage. In other words, the drive field couples exactly the same but with opposite polarity to each of the counter-wound coils, so that the voltages from the individual coils algebraically sum exactly to zero.
When an accumulation of magnetic particles on a solid substrate is placed in the gap in close proximity to the detection coils, the oscillating drive field produces a corresponding oscillating magnetization in the magnetic particles, which can then be detected by the detection coils. In the Simmonds patent, the physical size of the particle accumulations are closely matched to the size of one of the detection coils so that the accumulation of particles gives a signal in one coil at a time as the substrate is moved past the detection coil array. In this sense, one can think of the accumulation of magnetic particles as changing the effective balance of the detection coil array as the particle accumulation moves past the detection coils. As taught in Simmonds, the largest signal is obtained from any given accumulation of magnetic particles when the physical dimensions of the accumulation matches the dimensions of one of the individual coils in the detection coil array.
In the Simmonds implementation, the high frequency oscillating field (typically having a maximum amplitude of 500 to 1000 Oersted) serves two functions. First, it produces a large magnetization in the magnetic particles, and secondly, the high frequency nature of the oscillating field causes the induced magnetization of the magnetic particles to oscillate at the same frequency as the drive field (typically of order 100 KHz). Since the voltage induced in the inductive detection coils is proportional to the frequency of the detected signal, using a high frequency drive field with the inductive detection coils increases the sensitivity of the device and allows one to detect an extremely small quantity of magnetic particles.
A pending patent application related to the invention disclosed in Simmonds extends the basic concept in several ways (Ser. No. 09/451,660, filed Nov. 30, 1999, now U.S. Pat. No. 6,437,563 B1). First, it describes the use of an E-core design to provide higher symmetry in forming the gap, thereby allowing one to generate large drive fields while maintaining a high degree of balance with respect to the detection coils. Secondly, it discloses the use of alternative detection systems, including the use of other sensors such as fluxgate, giant magneto-resistance (GMR), colossal magneto-resistance (CMR), and Hall effect sensors, all still employing an AC drive field.
The detection system described by Simmonds exploits the fundamental magnetic behavior of the material comprising the magnetic particles to detect and measure their magnetization. The beads used in these applications are typically described as superparamagnetic, meaning that the beads are magnetic only when placed in an applied magnetic field. More specifically, they are not magnetic in the absence of an externally applied field, which is equivalent to saying that the beads have no (zero) remanent magnetization. In fact, beads used in biotechnology applications are rather carefully designed to have zero remanent magnetization because beads which do have a remanent magnetization stick together and clump up when placed in solution, causing a variety of problems. Hence, any device designed to detect or measure the magnetization of the beads typically used in biotechnology applications can work only if an external field is applied to the beads while the measurement is being performed. This situation should be distinguished from the techniques used in magnetic recording, where the magnetic film on the tape or disc is specifically designed to have a high remanent magnetization and a large coercive field. (The coercive field of a material gives the value of magnetic field that must be applied to completely demagnetize the material.) In fact, in recording applications the large remanent magnetization is the means by which information is actually stored.
The magnetic particles typically used in biotechnology applications are comprised of iron oxide, which is typically a mixture of Fe3O4 and Fe2O3, and measurements on particles from a variety of manufacturers have shown that the saturation magnetization of all these particles, regardless of their size, is about 500 Oersted. This is a very typical saturation field for these types of ferrite materials. To be more specific, this means that below about 500 Oersted, applying a larger field to the particles increases the magnetization of the particles, potentially increasing the signal to be detected. At fields above the saturation field, however, this effect is greatly reduced so that further increases in the applied magnetic field produce little or no increase in the magnetization of the particles. Furthermore, in virtually every magnetic detection system, noise sources exist that increase in proportion to the ambient magnetic field. Hence, increasing the applied field beyond the saturation level can actually degrade the measurement by decreasing its signal-to-noise ratio. The increase in the applied field can also make the undesired contribution to the signal from ferromagnetic contaminants more apparent. Hence, if one wishes to determine the number of particles present by measuring their magnetization, the optimal signal-to-noise ratio for such a measurement will normally be achieved by applying a magnetic field which is about the same as the saturation field of the particlesxe2x80x94in our case about 500 Oersted.
As discussed above, the Simmonds patent describes a technique for making quantitative measurements of superparamagnetic particles in bound complexes by applying a large oscillating magnetic field to the particles and detecting the oscillating magnetization induced in the particles. In this implementation, the sensors must be of the type which function well in the presence of a large oscillating magnetic field. The Simmonds apparatus is extremely sensitive and works very well. However, there are several factors which add complexity to the Simmonds design. Most importantly, because the Simmonds detection system works at a high frequency (typically about 100 KHz), there are capacitive coupling effects between the sample/substrate combination and the detection coils. In addition, such high frequency systems are always subject to phase shifts between the applied AC drive field and the detected signal. These effects can be very large when operating such a detection system at 100 KHz, and must be precisely accounted for if the system is to make an accurate measurement of the magnetization of the particles. Furthermore, generating the high frequency AC drive field in the gap of the toroidal magnetizer requires a significant amount of power, producing the possibility of thermal drifts in the detection electronics, especially immediately after the unit is first energized. The high power requirements of the AC drive field also impose a limitation on the length of time during which any such system can operate when running on battery power. And finally, the AC drive in the magnet and coil can potentially generate RF emissions which must be guarded against.
Broadly speaking, the present invention provides a greatly simplified and inexpensive method and apparatus for directly sensing and measuring very small accumulations of magnetic particles (for example, magnetite) and consequently, the bound complexes of interest.
A central feature of this invention is the use of a DC magnetic field (which replaces the time varying drive field in the prior art) to induce a magnetization in the magnetic particles, combined with the use of Hall sensors to detect the induced magnetization. The advantages of using a DC field instead of a time varying field are significant.
Generating a DC magnetic field in the implementation of this invention requires no field generating power source, is much simpler to implement than the prior AC driven system in the prior art, and can reduce the cost of the components by about two orders of magnitude. The requisite DC magnetic field can be generated without power consumption by using inexpensive permanent magnets and one or more pieces of iron to provide the appropriate field profile. In an exemplary prototype, the components used to produce the DC magnetic field cost less than about 25 cents. In contrast, the components used to generate the high frequency AC field used in previous devices cost in excess of twenty dollars and require significant power.
The power requirement to generate the 100 KHz AC field also limits the volume of the measurement region (the gap) in the devices which use an AC drive field, because the power required to generate the field increases with the volume of the gap. Increasing the ratio of area to height of the gap improves the field uniformity in the gap, but in systems which use AC drive fields this carries the penalty of increased power consumption. Increasing the volume of the gap when using a DC magnetic field requires only that correspondingly larger permanent magnets be used. A prototype system achieved fields in the order of 1000 Oersted in gaps having more than twice the volume of the Simmonds AC system.
The rather large magnetic field required to optimize these measurements (of order 500 Oersted) is incompatible with some types of sensors. However, the sensitivity of Hall sensors is not substantially degraded in high fields. In fact, Hall sensors can be designed to perform optimally in fields of this magnitude. The sensor area should also be matched to the sample size to maximize measurement sensitivity. While other types of sensors, including GMR and CMR sensors, can be engineered to meet the above criteria, Hall sensors matching these criteria are readily available and inexpensive.
A typical Hall sensor that might be used in this type of implementation is biased with approximately 10 to 20 milliamps of current. The output voltage of the sensor is proportional to both the applied field and the bias current. Hence, variations in the bias current will produce corresponding variations in the output signal, and it can be difficult electronically to produce extremely stable DC currents. This problem can be easily addressed, however, by applying an AC current to the Hall sensors, typically at a few kilohertz, which allows the generation of very stable peak amplitudes. Biasing the Hall sensors in this fashion also allows the detection system to work at a few kilohertz, thereby taking advantage of phase detection techniques to greatly improve the achievable signal-to-noise levels. (However, it should be understood that an AC current is not required in this invention, in which case there is simply a more stringent demand on the stability of the electronic circuitry that supplies the bias current for the sensors.)
A detection system using a DC magnetic field will also be largely immune to capacitive effects between the sample and the Hall sensors. In the high-frequency AC system of Simmonds, the dielectric properties in the sample substrate can cause significant capacitive coupling between the inductive detection coils, producing spurious signals. While these can be rejected using appropriate phase detection schemes, the DC system using Hall sensors is highly resistant to AC-coupled capacitive effects.
In the preferred embodiment, two individual Hall effect sensors are placed next to each other in an applied magnetic field, to form a matched pair of sensors. Each sensor produces a signal indicative of the magnetic field detected by the sensor. The signal from one sensor is subtracted from the other to form a resultant signal indicative of the difference in magnetic field in one sensor versus the signal in the other sensor. Performing this subtraction of signals electronically will significantly attenuate the unwanted resultant signal due to the applied field. Such a configuration of sensors is known as a xe2x80x9cgradiometerxe2x80x9d in the magnetic sensing industry.
In practice the measurement is performed by moving a well-defined pattern of magnetically susceptible particles past the two Hall sensors and in close proximity to them, while the particles are simultaneously exposed to the DC magnetic field. As taught in the Simmonds prior art, it is important to have the spatial dimensions of the pattern of magnetic particles closely match the physical dimensions of the Hall sensor. In this case, the pattern of magnetic particles is detected by the first Hall sensor as it moves past, and then after leaving the detection area of the first sensor, it is subsequently detected by the second Hall sensor. Since the two Hall sensors are connected such that they produce signals of opposite polarity, the difference signal between the two sensors is a function of the position of the spatial pattern as it moves past the two sensors, thereby indicating the number of particles present.